BackgroundIn vivo studies involving molecular markers of the follicle wall associated with follicular fluid (FF) milieu are crucial for a better understanding of follicle dynamics. The inability to obtain in vivo samples of antral follicle wall (granulosa and theca cells) without jeopardizing ovarian function has restricted advancement in knowledge of folliculogenesis in several species. The purpose of this study in mares was to develop and validate a novel, minimally invasive in vivo technique for simultaneous collection of follicle wall biopsy (FWB) and FF samples, and repeated collection from the same individual, during different stages of antral follicle development. We hypothesized that the in vivo FWB technique provides samples that maintain the normal histological tissue structure of the follicle wall layers, offers sufficient material for various cellular and molecular techniques, and allows simultaneous retrieval of FF.MethodsIn Experiment 1 (ex vivo), each follicle was sampled using two techniques: biopsy forceps and scalpel blade (control). In Experiment 2 (in vivo), FWB and FF samples from 10-, 20-, and 30-mm follicles were repeatedly and simultaneously obtained through transvaginal ultrasound-guided technique.ResultsIn Experiment 1, the thickness of granulosa, theca interna, and theca externa layers was not influenced (P > 0.05) by the harvesting techniques. In Experiment 2, the overall recovery rates of FWB and FF samples were 97 and 100%, respectively. However, the success rate of obtaining samples with all layers of the follicle wall and clear FF varied according to follicle size. The expression of luteinizing hormone receptor (LHR) was mostly confined in the theca interna layer, with the estradiol-related receptor alpha (ERRα) in the granulosa and theca interna layers. The 30-mm follicle group had greater (P < 0.05) LHR expression in the theca interna and ERRα in the granulosa layer compared to the other groups. The overall expression of LHR and ERRα, and the intrafollicular estradiol were higher (P < 0.05 – P < 0.0001) in the 30-mm follicle group.ConclusionThe in vivo technique developed in this study can be repeatedly and simultaneously used to provide sufficient FWB and FF samples for various cellular and molecular studies without jeopardizing the ovarian function, and has the potential to be translated to other species, including humans.Electronic supplementary materialThe online version of this article (10.1186/s12958-018-0380-8) contains supplementary material, which is available to authorized users.

Mathematical nutrition models have been developed for beef and dairy cattle to estimate dietary energy intake needed to change BCS. Similar technology has not been used to improve nutrition and feeding strategies for horses. An accurate equine nutrition model may enhance feeding management and reduce the costs of unnecessary overfeeding and promote an optimal level of fatness to achieve reproductive efficiency. The objectives of this study were to develop and evaluate a mathematical nutrition model capable of accurately predicting dietary energy changes to alter BW, rump fat (RF) thickness, and overall body fat (BF), which is needed to maximize profitability and productivity of mares. Model structure was similar to a previously developed model for cattle, and literature data for Quarter Horse mares were used to parameterize the horse model in predicting DE requirement associated with BCS changes. Evaluation of the horse model was performed using an independent dataset comprising 20 nonlactating Quarter Horse mares. Pretrial BCS was used to assign mares to 1 of 4 treatment groups and fed to alter BCS by 1 unit as follows: from 4 to 5 (Group 1), 5 to 4 (Group 2), 6 to 7 (Group 3), and 7 to 6 (Group 4). The BCS, RF thickness, and BW were measured for each mare before the commencement of the feeding trial and once per week thereafter for the duration of a 30-d feeding trial. Initial and target BCS, percent BF, and BW data were collected from each mare and inputted into the model. Mares were individually fed according to the DE suggestions proposed by the model to achieve the targeted BCS change within 30 d. The coefficient of determination of observed and model-predicted values (model precision) was 0.907 (P < 0.001) for BCS, 0.607 (P < 0.001) for percent BF, and 0.94 (P < 0.001) for BW. The BCS was highly correlated to percent BF (r = 0.808; P = 0.01). We concluded the reparameterized model was reliable to predict changes in BW and BCS, but more work is needed to improve the predictions of initial and final body composition.

Body condition score and percent body fat (BF; %) of horses are positively correlated with reproductive efficiency and are indicative of metabolic issues. However, BF in horses may be poorly predicted because current procedures are either subjective or dependent on one anatomical location. Therefore, the objectives of the current study were to compare 2 methods of predicting BF using rump fat thickness (RFT) and deuterium oxide (DO) dilution with actual tissue fat analysis by near-infrared spectroscopy (NIRS) in stock-type horses and to identify the relationship between BF and BCS. Twenty-four stock-type horses were selected to be humanely euthanized based on 3 primary criteria: geriatric, crippled, and/or unsafe. Approximately 20 h before slaughter, horses were weighed and BCS assessed to be 1 ( = 1; 433 kg), 2 ( = 1; 415 kg), 3 ( = 1; 376 kg), 4 ( = 7; 468 ± 13 kg), 5 ( = 10; 455 ± 11 kg), and 6 ( = 4; 493 ± 12 kg) and RFT was measured using ultrasonography. Blood samples were collected immediately before and 4 h after DO infusion (0.12 g/kg BW). Deuterium oxide concentration of plasma was determined by gas isotope ratio mass spectrometry. Horses were housed in a dry lot overnight before being individually sedated (1.1 mg xylazine/kg BW) and anesthetized using a jugular venipuncture (2.2 mg ketamine/kg BW), and potassium chloride (KCl) solution was administered to cease cardiac function before exsanguination. After euthanasia, horse carcasses were processed and dissected and tissues were collected for NIRS analysis. Body fat predicted by DO dilution was correlated with BF measured by NIRS analysis on various weight bases ( = 0.76 to 0.81, < 0.001), whereas no correlation was observed for BF predicted by RFT. A paired -test indicated that both DO dilution and RFT overestimated BF by 2.48 to 3.26% ( < 0.001) and 5.81 to 6.59% ( < 0.001), respectively, compared with NIRS analysis. Body condition scores affected BF measured by NIRS analysis ( ≤ 0.038) and predicted by DO dilution ( < 0.001) and RFT ( = 0.042). The current study provided evidence that DO dilution was a better prediction method for BF than RFT and that BCS may serve as a BF predictor in stock-type horses.

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SummaryReasons for performing study: Management decisions on unilateral orchiectomy are often influenced by the potential for post operative return to successful breeding. The effects of 2 surgical methods (first intention [FI] vs. second intention [SI] incision healing) for unilateral orchiectomy on resulting semen quality and scrotal temperature were evaluated. Objective: To evaluate the effects of 2 surgical unilateral orchiectomy techniques on scrotal healing, size of the remaining testis and post operative sperm quality. Materials and methods: Unilateral orchiectomy was performed on mature Miniature Horse stallions. Semen was collected prior to and up to 60 days after, unilateral orchiectomy. Semen parameters, scrotal and body temperatures, testis volume and days to incision healing were evaluated. Results: There was no effect of treatment or time on percent total sperm motility. Total sperm numbers were higher (P<0.05) 60 days after unilateral orchiectomy compared with 14 and 30 days. Percent viable sperm were higher (P<0.05) 30 and 60 days compared with pre-and 14 day post unilateral orchiectomy. Scrotal temperatures were lower after unilateral orchiectomy compared with preoperative values (PՅ0.003). Higher scrotal temperatures were recorded in Group FI, as compared with Group SI, during recovery from anaesthesia and at 1 and 2 h after surgery (P = 0.02). Mean time to incision healing was less in Group FI (10.0 days) than in Group SI (21.5 days; P = 0.05). Conclusion: In this study, total sperm motility was maintained and size of the remaining testis, total sperm numbers and percent viable sperm increased after unilateral orchiectomy. Incision healing time was shorter in Group FI; however, surgical technique did not have an effect on semen quality at 30 and 60 days post unilateral orchiectomy. Potential relevance: These data suggest that surgical technique for unilateral orchiectomy may not dramatically influence function of the remaining testis.